Eriksson, and Jeffrey F. D. Dean* ... bacteria (Dean and Eriksson, 1994). Although the .... ing. Proc Natl Acad Sci USA 88: 1247-1250. 1239-1243. 36: 41-66.
Plant Physiol. (1995) 107: 667-668
Planf Gene Regisfer
Nucleotide Sequence of a cDNA Clone Encoding an Acidic Laccase from Sycamore Maple (Acer pseudoplafanus L.)’ Peter R. LaFayette, Karl-Erik 1. Eriksson, and Jeffrey F. D. Dean* Department of Biochemistry and Molecular Biology, Center for Biological Resource Recovery, University of Georgia, Athens, Georgia 30602-7229
binding domains (Ohkawa et al., 1989) and the N-termina1 sequence of the purified protein (Sterjiades et al., 1992). To our knowledge, this represents the first complete sequence reported for a plant laccase, the character-
Laccases (p-diphenol:O, oxidoreductase, EC 1.10.3.2) are m,embers of a highly conserved class of metalloenzymes, the ”blue” copper oxidases, which includes ascorbate oxidase and ceruloplasmin (Rydén and Hunt, 1993). First identified more than 100 years ago in extracts of sap from the Japanese lacquer tree (Rhus vernicifera), laccases have since been identified in fungi, insects, higher plants, and bacteria (Dean and Eriksson, 1994). Although the physiological roles played by laccases in these various organisms are for the most part poorly understood, the enzymes generally oxidize diphenol or dinaphthol metabolites with subsequent reduction of O, to H,O. The oxidized aromatic products of these reactions often polymerize with each other or with molecules in the surrounding extracellular m a t r i x t o form chemically resilient structures that serve to protect the organism from various environmental stresses. Plant laccases were first proposed to play a role in lignin biosynthesis after Freudenberg et al. (1958) demonstrated that a fungal laccase could oxidize coniferyl alcohol in vitro with subsequent formation of a lignin-like dehydrogenation polymer. However, studies demonstrating that laccase purified from R. vernicifera could not oxidize coniferyl alcohol (Nakamura, 1967) and that laccase activity could not be detected histochemically in tissue sections taken from lignifying tree stems (Harkin and Obst, 1973) led to the conclusion that peroxidases, not laccases, catalyzed the final step in lignin deposition. More recent studies have suggested that such a conclusion was likely premature (Dean and Eriksson, 1994). Suspension-cultured cells of Acer psuedoplatanus secrete large quantities of a laccase capable of polymerizing lignin precursors (Sterjiades et al., 1992), and immunolocalization studies have shown that this enzyme is localized to the cell wall of lignifying vascular tissues in Acer stems (Driouich et al., 1992). A cDNA clone encoding this laccase was isolated through PCR-based screening (Israel, 1993) of a A phage library. Degenerate oligonucleotide probes were based on the amino acid sequence of conserved copper-
Table 1. Characteristics o f a cDNA clone encoding laccase from sycamore maple Organism: Sycamore maple (Acer pseudoplatanus L.). Function: Encodes laccase (pdiphenol:O, oxidoreductase, EC 1.1 0.3.2), an extracellular enzyme postulated to oxidize monolignols in the final step of lignin biosynthesis. Source: cDNA library in AUniZap XR vector constructed using poly(A+) mRNA isolated from suspension-cultured A. pseudoplatanus cells. Techniques: Clones were identified through a PCR-based library screening protocol using degenerate oligonucleotide primers derived from the published N-terminal protein sequence and copperbinding domains conserved in plant ascorbic acid oxidases and fungal laccasses. Clones were excised into pBluescript SK- in vivo, and cycle sequenced on both strands using a kit (TN-1000, Gold Biotechnology, St. Louis, MO) based on random insertion of a transposon harboring sequencing primer sites (Strathman et al., 1991). Method of Identification: Sequence identity of the deduced amino acid sequence with that determined for the N-terminal protein sequence of purified A. pseudoplatanus laccase. Features of cDNA: The clone was 2030 nucleotides in length and consisted of a 70-nucleotide 5’ untranslated region, a 1695-nucleotide open reading frame, and a 265-nucleotide 3‘ untranslated region. The 5’ untranslated region contained a stretch of 17 A’s 11 bases upstream of the translation start codon, and a polyadenylated tail was present at the 3’ end of the sequence. Features of Protein: A single open reading frame encoded a polypeptide of 565 amino acid residues having M, 62,600. Comparison with the N-terminal sequence of the purified laccase showed that a leader peptide of 23 amino acid residues having M, 2,700 preceded the mature protein of 542 amino acid residues having M, 59,900. The sequence contained 4 copper-binding domains and 16 potential N-linked glycosylation sites. Subcel Iular Location: Cell wall of lignifying tissues (Driouich et al., 1992).
This work was funded in part by U.S. Department of Energy grant No. DE-FG02-92ER20082 and by the Georgia Consortium for Technological Competitiveness in Pulp and Paper grant No. PP95FS3. * Corresponding author; e-mail jdeanx1Ouga.cc.uga.edu; fax 1-706-542-2222. 667
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istics of which are summarized in Table I. In a comparison with other blue copper oxidases found in GenBank (release 83.0) using BESTFIT (Genetic Computer Group Sequence Analysis software package, version 7.0), the complete A . psuedoplatanus laccase sequence was found to have 29% identity at the deduced amino acid leve1 with ascorbate oxidase from cucumber (Cucumis sativus) and 28% identity with the laccases cloned from Neuvospova crassa and Agaricus bisporous. In each case, most of the conserved amino acid residues were contained within the copper-binding domains. The cDNA was found to contain a stretch of 17 A's upstream of the predicted translation start site. The function of this sequence, if any, is unknown but could potentially be involved in poly(A)binding protein interactions.
ACKNOWLEDCMENT
We thank Dr. R. Sterjiades for contributions to this work.
Received August 24, 1994; accepted September 9, 1994. Copyright Clearance Center: 0032-0889/95/107/0667/02. The GenBank accession number for the sequence reported in this article is U12757.
Plant Physiol. Vol. 107, 1 9 9 5 LITERATURE ClTED
Dean JFD, Eriksson K-EL (1994) Laccase and the deposition of lignin in vascular plants. Holzforschung 48: 21-33 Driouich A, Lainé A-C, Vian B, Faye L (1992) Characterization and localization of laccase forms in stem and cell cultures of sycamore. Plant J 2: 13-24 Freudenberg K, Harkin JM, Rechert M, Fukuzumi T (1958)Die an der Verhozung beteiligten Enzyme. Die Dehydrierung des Sinapinalkohols. Chem Ber 91: 581-590 Harkin JM, Obst JR (1973) Lignification in trees: indication of exclusive peroxidase participation. Science 180 296-298 Israel DI (1993) A PCR-based method for high stringency screening of DNA libraries. Nucleic Acids Res 21: 2627-2631 Nakamura W (1967) Studies on the biosynthesis of lignin. I. Disproof against the catalytic activity of laccase in the oxidation of coniferyl alcohol. J Biochem 62: 54-61 Ohkawa J, Okada N, Shinmyo A, Takano M (1989) Primary structure of cucumber (Cucumis sutivus) ascorbate oxidase deduced from cDNA sequence: Homology with blue copper proteins and tissue-specific expression. Proc Natl Acad Sci USA 86: 1239-1243 Rydén LG, Hunt LT (1993) Evolution of protein complexity: the blue copper-containing oxidases and related proteins. J Mo1 Evol 36: 41-66 Sterjiades R, Dean JFD, Eriksson K-EL (1992) Laccase from sycamore maple (Acev pseudoplatnnus) polymerizes monolignols. Plant Physiol 99: 1162-1168 Strathman M, Hamilton BA, Mayeda CA, Simon MI, Meyerowitz EM, Palazzolo MJ (1991) Transposon-facilitated DNA sequencing. Proc Natl Acad Sci USA 88: 1247-1250
